1 Background

In this report, we will focus on studying the distances, Delta MES and MSR results of the splicing noise analysis of Ataxia RNAseq samples.

In this report, we will focus on studying the results of the mis-splicing noise analysis of Ataxia RNAseq samples: 46 Cerebellum and 45 Frontal Cortex samples. Samples will be pseudobulked by tissue, status disease and ataxia diagnosis, splitting the analysis in three different levels, including the results of the splicing noise between specific Ataxia diagnosis (i.e. SCA1, FRDA…) and control samples.

Changelog

v2.1

Changed the reference transcriptome to Gencode v38.

v2.0

Now, the clusters always compare the same number of samples between cases and controls. Procedure explained in methodology.
Added results for Level 2 (AtaxiaSubtype) and references.
Changed the reference transcriptome from Gencode v43 to Gencode v39.
Updated methodology.

v1.1

Updated subsampling method to consider other covariates other than RIN. Each covariate was weighted to contribute according to its influence to the number of novel junctions for each sample. For both tissues, RIN was the most relevant contributor with more than 85% of the weight. Thus, no relevant variation from the previous approach was observed.

2 Methods

2.1 Analysis pipeline (splicing noise evaluation)

For every available BAM file to study, we apply the following steps:

Download and extraction of BAM files: the files are downloaded from s3://ataxia-bulk-rnaseq/nextflow_first_attemp/Star_2_pass_by_indv/output/STAR/align/BAM_files/ and subfolders.
Junction extraction: all junctions are extracted using regtools junction extract after sorting and indexing with samtools. A file is created for each BAM file in BED12 format.
Junction annotation: the junctions are read from the previously created files and merged into a single dataframe of read junctions. We also register the number of reads of each junction in every sample. The junctions located within the ENCODE blacklisted regions v2 are removed. The junction_annot() function from the package dasper is used to annotate the junctions to the Gencode v38 reference transcriptome. All junctions not classified as either novel_donor, novel_acceptor or annotated are ignored. We also remove all junctions smaller than 25bp (base pairs) and annotated introns that are ambiguously assigned to more than one gene.
Junction pairing: by looking for overlaps between the novel junctions and the annotated junctions for each sample, we measure the distance in bp between the novel and reference splice site. The annotated introns that are never associated to a novel junction are considered a never misspliced junctions.
Filtering the distances: we remove the pairings in which a novel junctions are associated to more than one reference intron across different samples. For more information about this process, please see the methods section in Introverse paper [1].

Next, we need to decide on a clustering method to combine and compare different samples. More information in section about clustering.

Measuring the mis-splicing ratio: by adding all novel junction read counts attached to an annotated intron across all samples in which the novel splice was observed, and then dividing by the total number of reads of the annotated intron and the novel junctions across the same set of samples, we obtain a measurement of the mis-splicing ratio for an given annotated intron at both the donor splice site and the acceptor splice site. For more information about the mis-splicing ratio, please see section MSR.
Generation of the DB: two tables are created per each cluster: db_introns and db_novel. Each one contains the relevant information related to reference introns (including the never misspliced introns) and novel junctions. This includes the MaxEntScan scores, the percentage of protein-coding transcripts and the classification in u2 and u12 introns.

2.2 Clustering (pseudobulk)

In our dataset, we have a total of 95 samples, corresponding to 48 different individuals. A total of 4 samples are removed because they belong to individuals diagnosed as CANVAS and AIFM1.

Three different level of studies were studied in this report, and always different analyses for each tissue.

Level 1 (Type): whether the sample is diagnosed with ataxia or not (diases status). For the frontal cortex tissue, samples with \(RIN<4\) will be removed, while for the Cerebellum tissue only controls with \(RIN<=7\) are kept. This is to ensure non-significant differences in the RIN medians.
Level 2 (AtaxiaSubtype): two different analyses are performed: known ataxia cases vs. controls and unknown ataxia cases vs. controls. In both scenarios, no restrictions about RIN is required.
Level 3 (Diagnosis): a different analysis was performed for every ataxia diagnosis with at least three samples: FRDA, SCA1, SCA2 and SCA6. In all situations, control samples are selected to minimize a weighted Gower distance to the case samples (more information in following section).

2.3 Subsampling

Studies about the relationship in mis-splicing ratio’s median and the number of samples pseudobulked showed a clear correlation between the two. In order to avoid this effect in the comparisons between cases and controls, we decided to subsample the majority class until both classes have the same number of samples. The subsampling was performed so that the weighted Gower distance between the case samples and the control samples was minimized.

For datasets with both quantitative (i.e. RIN) and categorical (i.e. Brain bank) variables, also called mixed datasets, Gower’s distance is a common measurement of similarity between any two samples [2]. The similarity between samples can be defined as:

\[ S_{ij}=\frac{\sum_{k=1}^p s_{ijk}\delta_{ijk}w_k}{\sum_{k=1}^p \delta_{ijk}w_k} \] where \(p\) are the variables beeing compared, \(i\) and \(j\) refers to two different samples and:

Quantitative variables: \(s_{ijk}=1-|x_{ik}-x_{jk}|/R_k\) where \(R_k\) is the range of the variable \(k\).
Categorical variables: \(s_{ijk}=1\) if \(x_{ik}=x_{jk}\) and \(s_{ijk}=0\) otherwise.
\(\delta_{ijk}\): whether a comparison between sample \(i\) and sample \(j\) can be performed for variable \(k\).
\(w_k\): optional weight to increase or decrease the relevance of certain variables.

We decided to apply a weighting to the variables in order to increase the relevance of the main contributors to the number of junction reads across samples.

The subsampling process was the following:

Variable weights: first, we divided the samples by tissue. Then, for each sample, we extract the number of reads associated to both annotated and novel junctions. Using a linear model to predict this number of reads, we measure the variance explained by each of the main covariates that will be employed in subsampling: RIN, PMI, Age at death, Brain bank and sex. The percentage of variance explained by each covariate will be considered as the weight in the next step.
Gower’s distance between samples: for each the minority class sample, the most similar majority class sample was selected without repetition (i.e. once two samples were assigned together, none of them can be selected again). Thus, the same number of samples between the two classes were obtained.
Wilcoxon test: between the two sets of samples, a Wilcoxon test is executed to test whether there are significant differences in the RIN medians. If significant differences are found, some extra restriction is applied to any of the sets. For example, for Cerebellum Level 1 study, control samples with RIN higher than 7 needed to be removed to ensure non-significant differences in the median.

The obtained weights are the following:

Covariate	Cerebellum weights	Frontal Cortex weights
RIN	0.989	0.855
PMI	0.001	0.132
Brain Bank	0.003	0.013
Age at death	0.001	0.000
Sex	0.006	0.000

2.4 Common annotated introns

For each study, we will only consider the common annotated introns between the two classes. To do so, we generate the following dataframes:

Common annotated intron table: we looped through both db_introns tables and extracted only the information from the common annotated introns in the clusters. To identify common annotated introns, we used their locus (i.e. seqname:start-end:strand), since it is a unique identifier. The goal is to have the same number of annotated between cases and controls.
Common novel junction table: we looped through both db_novel tables and extracted only the information from the novel junctions associated to common annotated introns. Thus, we first needed to calculate the common annotated intron table.

3 Studied metrics

The examples in this section corresponds to the Level 1 study for Cerebellum.

Distances

The distances graph is generated by counting the number of unique novel junctions at any given distance of the reference splicing site. From the list of novel junctions associated to common reference introns, we group by novel_type and cluster, count the number of entries for each distance within 30 bp into both intron and exon sequence, and represent the data in a histogram. An example of the data can be seen in the following table:

Novel jun. ID	Novel type	Cluster	Sequence	Distance [bp]
1900408	novel_acceptor	Control	intron	-26
262172	novel_acceptor	Case	exon	4
1562780	novel_acceptor	Case	exon	75
1744154	novel_acceptor	Control	exon	26
2006682	novel_acceptor	Case	exon	84
315218	novel_donor	Control	exon	3
1123582	novel_acceptor	Control	intron	-22
149464	novel_acceptor	Case	exon	89
951814	novel_donor	Case	exon	10
446245	novel_acceptor	Case	exon	29

For each study, different graphical representations will be presented. First, we present a histogram of the different distance values between -30 and 30 bp, with the case bars stacked on top of the control bars:

However, this representation does not allow for an easy comparison between cases and controls because of the bar stacking. To do so, we split the graph in two more facets to represent each cluster by its own:

If we focus on the difference in the number of unique novel junctions between cases and controls, the last graph represent the histogram resulting from subtracting the control distances from the case distances:

Distances (modulo 3)

To generate the modulo 3 graphs, we first filter by those distances smaller than 100 bp in either direction. Then, we group by novel_type and cluster, and calculate the distance in modulo 3 (i.e. the remainder from the division of the distance by 3). Once we have all novel junctions split in the four different categories with their distances in modulo 3, we calculate the percentage that each modulo 3 represents in each category. Thus, for each category:

\[ \text{mod.0 }\% = \frac{\# 0}{\#0 + \#1 + \#2}*100\%\qquad\quad \text{mod.1 }\% = \frac{\# 1}{\#0 + \#1 + \#2}*100\%\qquad\quad \text{mod.2 }\% = \frac{\# 2}{\#0 + \#1 + \#2}*100\% \]

Here, \(\#X\) represents the number of novel junctions at a modulo 3 distance equal to \(X\). For example, from the information of the following table, we can focus on the Case acceptor group, where we observe two 0s, two 1s and one 2:

Novel jun. ID	Novel type	Cluster	Sequence	Distance [bp]	Mod3 distance [bp]
Case acceptor
262172	novel_acceptor	Case	exon	4	1
1562780	novel_acceptor	Case	exon	75	0
2006682	novel_acceptor	Case	exon	84	0
149464	novel_acceptor	Case	exon	89	2
446245	novel_acceptor	Case	exon	29	2
Case donor
951814	novel_donor	Case	exon	10	1
Control acceptor
1900408	novel_acceptor	Control	intron	-26	2
1744154	novel_acceptor	Control	exon	26	2
1123582	novel_acceptor	Control	intron	-22	1
Control donor
315218	novel_donor	Control	exon	3	0

Thus, the calculated percentage are:

\[ \text{mod.0 }\% = 40\%\qquad\quad \text{mod.1 }\% = 40\%\qquad\quad \text{mod.2 }\% = 20\% \]

Additionally, we can further group the novel junctions by their intron/exon sequence, resulting a total of 8 categories, where the same calculation of percentages can be applied:

Novel jun. ID	Novel type	Cluster	Sequence	Distance [bp]	Mod3 distance [bp]
Case acceptor exon
262172	novel_acceptor	Case	exon	4	1
1562780	novel_acceptor	Case	exon	75	0
2006682	novel_acceptor	Case	exon	84	0
149464	novel_acceptor	Case	exon	89	2
446245	novel_acceptor	Case	exon	29	2
Case donor exon
951814	novel_donor	Case	exon	10	1
Control acceptor exon
1744154	novel_acceptor	Control	exon	26	2
Control acceptor intron
1900408	novel_acceptor	Control	intron	-26	2
1123582	novel_acceptor	Control	intron	-22	1
Control donor exon
315218	novel_donor	Control	exon	3	0

With the previous tables in mind, we represent them in three different ways. The first one just represents the number of unique novel junctions for each modulo 3 distance where \(abs(distance) <= 100\).

We also represent the percentage that each that each distance in modulo 3 represents when grouped by novel_type (i.e. acceptor/donor) and cluster (i.e. case/control).

Lastly, we represent the same data as before but taking into consideration whether it is located in the intronic or exonic sequence:

Delta MaxEntScan Score

To represent the difference in MaxEntScan scores (MES), we subtract the MaxEntScan score of the annotated intron to the MaxEntScan score of a novel junction at each splice site. We obtain a table like the following, and represent the density plot of the different values for delta_ss5score and delta_ss3score:

\[ \text{Delta MES}^{5'} = \text{MES}_{ref}^{5'}-\text{MES}_{novel}^{5'}\qquad\qquad\text{Delta MES}^{3'} = \text{MES}_{ref}^{3'}-\text{MES}_{novel}^{3'} \]

Example table with the Delta MES data:

Novel jun. ID	Ref. jun. ID	Delta ss5score	Delta ss3score
2035214	2035210	2.63	3.96
89328	89327	6.31	-0.30
67502	67507	3.53	-0.88
381357	381360	-2.17	0.00
1547859	1547861	2.25	1.54
1151075	1151074	-0.76	0.00
1877181	1877182	22.15	22.18
704572	704556	4.77	11.05
1533950	1533949	2.09	1.97
806079	806085	0.05	0.00

As for the visual representations, the difference between the MaxEntScan scores for each splice site is shown in the X-axis. The Y-axis represents the kernel density estimate (smoothed version of a histogram). The fill color represents cases vs. controls.

Additionally, we can also represent the difference in the kernel density estimate between case and control:

MSR

The Mis-Splicing Ratio (MSR) is calculated in the data-analysis pipeline for each annotated intron found in the project samples. More information about the mis-splicing ratio can be found in the Ataxia RNAseq Pseudobulk - Median MSR Analaysis report. To visualize this data, we represent the density plot for MSR at the different splice sites (i.e. donor or acceptor). Example table with the MSR data:

Ref. jun, ID	Cluster	Ref. type	MSR Donor	MSR Acceptor
1331421	Control	never	0.000	0.000
1972189	Control	both	0.059	0.004
414325	Case	acceptor	0.000	0.033
750187	Control	never	0.000	0.000
1996411	Case	never	0.000	0.000
573131	Case	donor	0.004	0.000
253464	Case	never	0.000	0.000
448971	Control	never	0.000	0.000
403929	Case	never	0.000	0.000
1503717	Control	never	0.000	0.000

We represent both the distribution of mis-splicing ratio (MSR) at the donor site and the acceptor site. The X-axis represents the MSR, while the Y-axis represents the kernel density estimate.

## <br><br><br>

Note that the distributions have a discontinuity at \(x=0.05\) to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis will also be present if the difference between the two sample types exceeds a predefined threshold.

Stats

Under the Stats subsection, we will study different statistics of the annotated introns and novel junctions:

Unique annotated introns: number of annotated introns classified as never mis-spliced, mis-spliced at acceptor end, donor end or both. It is represented by cluster, so that we can study the variation in the number and percentage of never mis-spliced annotated introns.
Reads - annotated introns: total reads associated to annotated introns and the percentage that they represent against the total read depth.
Reads - novel junctions: total reads associated to novel junctions and the percentage that they represent against the total read depth. Results are separated by cluster.

4 Results for Level 1 (Type)

4.1 Cerebellum

Distribution of sample RIN for Cerebellum level 1 study:

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Case	198167	40435	20.40
acceptor	Control	198167	39710	20.04
both	Case	198167	33845	17.08
both	Control	198167	34144	17.23
donor	Case	198167	21951	11.08
donor	Control	198167	21934	11.07
never	Case	198167	101936	51.44
never	Control	198167	102379	51.66

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	198167	4052024612	423466847	10.45
Control	198167	4520672127	521789300	11.54

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	234748	136989	4052024612	2355486	0.06
Case	novel_donor	234748	97759	4052024612	1700082	0.04
Control	novel_acceptor	238281	137983	4520672127	2803847	0.06
Control	novel_donor	238281	100298	4520672127	2003931	0.04

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

4.2 Frontal Cortex

Distribution of sample RIN for Frontal Cortex level 1 study:

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Case	212490	42989	20.23
acceptor	Control	212490	42227	19.87
both	Case	212490	38202	17.98
both	Control	212490	37676	17.73
donor	Case	212490	25739	12.11
donor	Control	212490	25323	11.92
never	Case	212490	105560	49.68
never	Control	212490	107264	50.48

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	212490	4769291070	854565112	17.92
Control	212490	5300507311	862005120	16.26

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	270355	152012	4769291070	2948776	0.06
Case	novel_donor	270355	118343	4769291070	2176172	0.05
Control	novel_acceptor	265070	147966	5300507311	3030018	0.06
Control	novel_donor	265070	117104	5300507311	2263035	0.04

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

5 Results for Level 2 (AtaxiaSubtype)

5.1 Cerebellum

Distribution of sample RIN for Cerebellum level 2 study:

KnownAtaxia

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	195965	38514	19.65
acceptor	KnownAtaxia	195965	36451	18.60
both	Control	195965	31939	16.30
both	KnownAtaxia	195965	27608	14.09
donor	Control	195965	21431	10.94
donor	KnownAtaxia	195965	21444	10.94
never	Control	195965	104081	53.11
never	KnownAtaxia	195965	110462	56.37

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	195965	3448881421	324372917	9.41
Control	195965	3977174051	432584614	10.88

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	189450	108616	3448881421	1741540	0.05
Case	novel_donor	189450	80834	3448881421	1260992	0.04
Control	novel_acceptor	221459	128194	3977174051	2379406	0.06
Control	novel_donor	221459	93265	3977174051	1688372	0.04

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

UnknownAtaxia

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	182348	26187	14.36
acceptor	UnknownAtaxia	182348	31827	17.45
both	Control	182348	14442	7.92
both	UnknownAtaxia	182348	19905	10.92
donor	Control	182348	16653	9.13
donor	UnknownAtaxia	182348	17614	9.66
never	Control	182348	125066	68.59
never	UnknownAtaxia	182348	113002	61.97

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	182348	1723566342	159025954	9.23
Control	182348	1693165552	136668669	8.07

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	137582	80978	1723566342	856784	0.05
Case	novel_donor	137582	56604	1723566342	612918	0.04
Control	novel_acceptor	102863	58382	1693165552	669761	0.04
Control	novel_donor	102863	44481	1693165552	458738	0.03

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

5.2 Frontal Cortex

Distribution of sample RIN for Frontal Cortex level 2 study:

KnownAtaxia

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	209708	40473	19.30
acceptor	KnownAtaxia	209708	41017	19.56
both	Control	209708	34187	16.30
both	KnownAtaxia	209708	34357	16.38
donor	Control	209708	24851	11.85
donor	KnownAtaxia	209708	25050	11.95
never	Control	209708	110197	52.55
never	KnownAtaxia	209708	109284	52.11

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	209708	3572908572	665252255	18.62
Control	209708	4100844167	702866511	17.14

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	241427	134997	3572908572	2283057	0.06
Case	novel_donor	241427	106430	3572908572	1709242	0.05
Control	novel_acceptor	239218	133056	4100844167	2464214	0.06
Control	novel_donor	239218	106162	4100844167	1849022	0.05

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

UnknownAtaxia

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	195328	31309	16.03
acceptor	UnknownAtaxia	195328	30651	15.69
both	Control	195328	19848	10.16
both	UnknownAtaxia	195328	17533	8.98
donor	Control	195328	20335	10.41
donor	UnknownAtaxia	195328	19693	10.08
never	Control	195328	123836	63.40
never	UnknownAtaxia	195328	127451	65.25

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	195328	1659144178	253102013	15.25
Control	195328	1765693058	293450631	16.62

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	126819	71270	1659144178	833338	0.05
Case	novel_donor	126819	55549	1659144178	618577	0.04
Control	novel_acceptor	140907	78017	1765693058	972618	0.06
Control	novel_donor	140907	62890	1765693058	760132	0.04

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

6 Results for Level 3 (Diagnosis)

6.1 Cerebellum

Distribution of sample RIN for Cerebellum level 3 study:

FRDA

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	176181	23705	13.45
acceptor	FRDA	176181	24542	13.93
both	Control	176181	12569	7.13
both	FRDA	176181	13221	7.50
donor	Control	176181	15157	8.60
donor	FRDA	176181	15665	8.89
never	Control	176181	124750	70.81
never	FRDA	176181	122753	69.67

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	176181	896665067	89994863	10.04
Control	176181	937441018	94447687	10.08

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	93579	53271	896665067	527632	0.06
Case	novel_donor	93579	40308	896665067	377082	0.04
Control	novel_acceptor	89193	50708	937441018	508712	0.05
Control	novel_donor	89193	38485	937441018	349410	0.04

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA1

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	168115	19582	11.65
acceptor	SCA1	168115	18882	11.23
both	Control	168115	9200	5.47
both	SCA1	168115	8590	5.11
donor	Control	168115	12736	7.58
donor	SCA1	168115	13153	7.82
never	Control	168115	126597	75.30
never	SCA1	168115	127490	75.83

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	168115	663185400	55447132	8.36
Control	168115	727022405	60692971	8.35

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	64141	35767	663185400	281691	0.04
Case	novel_donor	64141	28374	663185400	210850	0.03
Control	novel_acceptor	67003	38156	727022405	327377	0.05
Control	novel_donor	67003	28847	727022405	233144	0.03

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA2

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	159886	13505	8.45
acceptor	SCA2	159886	14135	8.84
both	Control	159886	4523	2.83
both	SCA2	159886	4381	2.74
donor	Control	159886	9123	5.71
donor	SCA2	159886	9079	5.68
never	Control	159886	132735	83.02
never	SCA2	159886	132291	82.74

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	159886	584147295	38935749	6.67
Control	159886	710683525	39604626	5.57

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	39416	22961	584147295	182180	0.03
Case	novel_donor	39416	16455	584147295	132288	0.02
Control	novel_acceptor	38531	21928	710683525	169624	0.02
Control	novel_donor	38531	16603	710683525	118165	0.02

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA6

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	170971	20916	12.23
acceptor	SCA6	170971	19858	11.61
both	Control	170971	10418	6.09
both	SCA6	170971	8296	4.85
donor	Control	170971	13521	7.91
donor	SCA6	170971	12953	7.58
never	Control	170971	126116	73.76
never	SCA6	170971	129864	75.96

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	170971	616156554	67782298	11.00
Control	170971	712278697	68984361	9.69

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	64023	36427	616156554	316179	0.05
Case	novel_donor	64023	27596	616156554	220631	0.04
Control	novel_acceptor	74679	42471	712278697	403422	0.06
Control	novel_donor	74679	32208	712278697	275237	0.04

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

6.2 Frontal Cortex

Distribution of sample RIN for Frontal Cortex level 3 study:

FRDA

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	190016	28043	14.76
acceptor	FRDA	190016	26193	13.78
both	Control	190016	16142	8.50
both	FRDA	190016	13383	7.04
donor	Control	190016	18596	9.79
donor	FRDA	190016	17677	9.30
never	Control	190016	127235	66.96
never	FRDA	190016	132763	69.87

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	190016	866370018	155141061	17.91
Control	190016	1043087110	193646726	18.56

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	100061	55394	866370018	538635	0.06
Case	novel_donor	100061	44667	866370018	402906	0.05
Control	novel_acceptor	115374	63869	1043087110	696453	0.07
Control	novel_donor	115374	51505	1043087110	503840	0.05

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA1

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	185853	25038	13.47
acceptor	SCA1	185853	23801	12.81
both	Control	185853	12990	6.99
both	SCA1	185853	11645	6.27
donor	Control	185853	17029	9.16
donor	SCA1	185853	16604	8.93
never	Control	185853	130796	70.38
never	SCA1	185853	133803	71.99

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	185853	698071292	143266471	20.52
Control	185853	785114845	148634904	18.93

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	88195	48508	698071292	453261	0.06
Case	novel_donor	88195	39687	698071292	345121	0.05
Control	novel_acceptor	95396	52532	785114845	486387	0.06
Control	novel_donor	95396	42864	785114845	370534	0.05

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA2

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	183050	24450	13.36
acceptor	SCA2	183050	22490	12.29
both	Control	183050	12391	6.77
both	SCA2	183050	9805	5.36
donor	Control	183050	16802	9.18
donor	SCA2	183050	14962	8.17
never	Control	183050	129407	70.69
never	SCA2	183050	135793	74.18

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	183050	645767566	117766633	18.24
Control	183050	762434189	141342187	18.54

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	77025	43272	645767566	380720	0.06
Case	novel_donor	77025	33753	645767566	283002	0.04
Control	novel_acceptor	92338	50811	762434189	474444	0.06
Control	novel_donor	92338	41527	762434189	368842	0.05

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA6

Distances (N)

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions. Bars are stacked.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Number of unique novel junctions.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details

X-Axis: Distance between the novel splice site and the reference splice site.
Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details

X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
Colour: Cluster type as case vs. control.
Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Kernel density estimate (smoothed version of a histogram).
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

Expand details

X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
Y-Axis: Difference between the kernel density estimate for case samples and control samples.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.

MSR

## <br><br><br>

Expand details

X-Axis: The mis-splicing ratio.
Y-Axis: Kernel density estimate.
Colour: Cluster type as case vs. control.
Facets: Different splice sites.
Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site	Cluster	# Annotated introns	# Ann. introns by splice site	Percent [%]
acceptor	Control	183595	23996	13.07
acceptor	SCA6	183595	24755	13.48
both	Control	183595	11435	6.23
both	SCA6	183595	13080	7.12
donor	Control	183595	16185	8.82
donor	SCA6	183595	16434	8.95
never	Control	183595	131979	71.89
never	SCA6	183595	129326	70.44

Expand details

# Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:

\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster	# Annotated introns	# Reads in cluster	# Ann. intron reads	Percentage [%]
Case	183595	669517733	120694740	18.03
Control	183595	782827004	126120311	16.11

Expand details

# Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster	Novel type	# Novel junctions	# Novel junc. in splice site	# Reads in cluster	# Novel junc. reads	Percentage [%]
Case	novel_acceptor	93862	52528	669517733	423931	0.06
Case	novel_donor	93862	41334	669517733	318321	0.05
Control	novel_acceptor	85458	47409	782827004	418368	0.05
Control	novel_donor	85458	38049	782827004	317316	0.04

Expand details

# Novel junctions: total number of novel junctions for that particular cluster.
# Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
# Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
# Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.

\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

7 Session info

Show/hide

## ─ Session info ───────────────────────────────────────────────────────────────────────────────────────────────────────
##  setting  value
##  version  R version 4.2.1 (2022-06-23)
##  os       Ubuntu 20.04.4 LTS
##  system   x86_64, linux-gnu
##  ui       X11
##  language (EN)
##  collate  en_US.UTF-8
##  ctype    en_US.UTF-8
##  tz       Etc/UTC
##  date     2023-06-05
##  pandoc   2.18 @ /usr/lib/rstudio-server/bin/quarto/bin/tools/ (via rmarkdown)
## 
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8 References

[1]

Garcı́a-Ruiz S, Zhang D, Gustavsson EK, Rocamora-Perez G, Grant-Peters M, Fairbrother-Browne A, et al. Splicing accuracy varies across human introns, tissues and age 2023. https://doi.org/10.1101/2023.03.29.534370.

[2]

Hummel M, Edelmann D, Kopp-Schneider A. Clustering of samples and variables with mixed-type data. PLOS ONE 2017;12:e0188274. https://doi.org/10.1371/journal.pone.0188274.

Ataxia RNAseq - Distances, deltaMES and MSR distribution Analysis

Guillermo Rocamora Pérez

05 June 2023

1 Background

Changelog

v2.1

v2.0

v1.1

2 Methods

2.1 Analysis pipeline (splicing noise evaluation)

2.2 Clustering (pseudobulk)

2.3 Subsampling

2.4 Common annotated introns

3 Studied metrics

Distances

Distances (modulo 3)

Delta MaxEntScan Score

MSR

Stats

4 Results for Level 1 (Type)

4.1 Cerebellum

Distances (N)

Modulo 3

Delta MaxEntScan

MSR

Stats

Unique annotated introns

Reads - annotated introns

Reads - novel junctions

4.2 Frontal Cortex

Distances (N)

Modulo 3

Delta MaxEntScan

MSR

Stats

Unique annotated introns

Reads - annotated introns

Reads - novel junctions

5 Results for Level 2 (AtaxiaSubtype)

5.1 Cerebellum

KnownAtaxia

Distances (N)

Modulo 3

Delta MaxEntScan

MSR

Stats

Unique annotated introns

Reads - annotated introns

Reads - novel junctions

UnknownAtaxia

Distances (N)

Modulo 3

Delta MaxEntScan

MSR

Stats

Unique annotated introns

Reads - annotated introns

Reads - novel junctions

5.2 Frontal Cortex

KnownAtaxia

Distances (N)

Modulo 3

Delta MaxEntScan

MSR

Stats

Unique annotated introns

Reads - annotated introns

Reads - novel junctions

UnknownAtaxia

Distances (N)

Modulo 3

Delta MaxEntScan

MSR

Stats

Unique annotated introns

Reads - annotated introns

Reads - novel junctions

6 Results for Level 3 (Diagnosis)

6.1 Cerebellum

FRDA